Soluble TNFR1 was originally characterized as a proteolytically cleaved receptor ectodomain that is released by a receptor sheddase. This project has identified several new regulatory mechanisms for generation of soluble cytokine receptors that do not involve the proteolytic cleavage of receptor ectodomains. First, we hypothesized the existence of regulatory proteins that modulate TNFR1 release to the extracellular compartment. Utilizing a yeast-two hybrid approach, we identified ARTS-1 (Aminopeptidase Regulator of TNF Receptor Shedding) as a type II integral membrane protein that binds the full-length 55-kDa TNFR1 and promotes TNFR1 release from human airway and vascular endothelial cells (HUVEC) (JCI 2002;110: 515-526). Second, we showed that HUVEC constitutively release TNFR1 to the extracellular compartment primarily as a full-length, 55-kDa protein (PNAS 2004;101: 1297-302). This finding lead to the discovery that full-length TNFR1 is released within the membranes of exosome-like vesicles via a zinc metalloprotease-dependent process that does not involve receptor sheddase activity. Thus, the release of TNFR1 exosome-like vesicles represents a novel, alternative mechanism for the release of cytokine receptors from cells that is distinct from the proteolytic cleavage of receptor ectodomains or the generation of alternatively spliced translation products(J Immunology 2004;173: 5343-8). The physiological relevance of these observations was confirmed by the demonstration in human subjects of TNFR1 exosome-like vesicles in serum and bronchoalveolar lining fluid. Third, we showed that ARTS-1 promotes the release of soluble, cleaved forms of IL-6Ra (J Biol Chem 2003;278: 28677-85) and IL-1RII (J Immunology 2003;171: 6814-9). Thus, ARTS-1 regulates the release of three distinct cytokine receptor superfamilies, the TNF receptor superfamily (TNFR1), the class I cytokine receptor superfamily (IL-6Ra), and the immunoglobulin/Toll-like receptor superfamily (IL-1RII). We have also identified nucleobindin 2 (NUCB2, NEFA) as a calcium-dependent, ARTS-1-binding protein that associates with intracytoplasmic TNFR1 vesicles and is required for the constitutive release of TNFR1 within the membranes of exosome-like vesicles, as well the IL-1b-mediated, inducible proteolytic cleavage of TNFR1 (JBC 2006;281: 6860-6873). Therefore, NUCB2 and ARTS-1 regulate two zinc metalloprotease-dependent mechanisms of cytokine receptor shedding, the sheddase-independent, constitutive release of exosome-like vesicles containing full-length TNFR1 receptors and the sheddase-dependent, inducible proteolytic cleavage of receptor ectodomains. This project has identified several new insights regarding the release of TNFR1 to the extracellular space: I. The regulation of TNFR1 release pathways appears to involve the trafficking of cytoplasmic TNFR1 vesicles. Vesicular trafficking is controlled by ADP-ribosylation factors (ARFs), which are active in the GTP-bound state and inactive when bound to GDP. ARF activation is enhanced by guanine nucleotide-exchange factors that catalyze replacement of GDP by GTP. We investigated whether the brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins, BIG1 and/or BIG2, are required for TNFR1 release from HUVEC. RNA interference (RNAi) showed that BIG2, but not BIG1, regulated the release of TNFR1 exosome-like vesicles, whereas neither BIG2 nor BIG1 was required for the IL-1b-induced proteolytic cleavage of TNFR1 ectodomains. BIG2 co-localized with TNFR1 in cytoplasmic vesicles and the association between BIG2 and TNFR1 was disrupted by BFA. Consistent with the preferential activation of class I ARFs by BIG2, ARF1 and ARF3 participated in the extracellular release of TNFR1 exosome-like vesicles in a non-redundant and additive fashion. Thus, we identified that the association between BIG2 and TNFR1 selectively regulates the extracellular release of TNFR1 exosome-like vesicles via an ARF1- and ARF3-dependent mechanism, but did not affect the inducible proteolytic cleavage of TNFR1 ectodomains (JBC 2007;282: 9591 - 9599). II. BIG2 contains three A kinase-anchoring protein (AKAP) domains that may coordinate cAMP and ARF regulatory functions. We hypothesized that BIG2 might regulate the release of TNFR1 exosome-like vesicles via its AKAP, as well as its Sec7 domains. We showed that 8-Br-cAMP induced the release of full-length, 55-kDa TNFR1 within exosome-like vesicles via a PKA-dependent mechanism. RNA interference experiments showed that RIIb modulates both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles. Consistent with its AKAP function, BIG2 was required for the cAMP-induced PKA-dependent release of TNFR1 exosome-like vesicles via a mechanism that involved the binding of RIIb to BIG2 AKAP domains B and C. This showed that both the constitutive and cAMP-induced release of TNFR1 exosome-like vesicles occur via PKA-dependent pathways that are regulated by the anchoring of RIIb to BIG2 via AKAP domains B and C. Thus, BIG2 regulates TNFR1 exosome-like vesicle release by two distinct mechanisms, as a guanine nucleotide-exchange protein that activates class I ARFs and as an AKAP for RIIb that localizes PKA signaling within cellular TNFR1 trafficking pathways (JBC 2008;283: 25364-71). III. Co-immunoprecipitation experiments identified RBMX (RNA-binding motif gene, X chromosome)as an ARTS-1-associated protein that regulates both the constitutive release of TNFR1 exosome-like vesicles and the inducible proteolytic cleavage of TNFR1 ectodomains (BBRC 2008;371: 505-9). IV. Since TNFR1 exosome-like nanovesicles are released by HUVEC, we hypothesized that they may circulate in human blood and modulate TNF-mediated inflammation. TNFR1 exosome-like vesicles were demonstrated in human serum by immunoelectron microscopy. Western blots of human plasma showed a 48-kDa TNFR1, which is consistent with a membrane-associated receptor. Gel exclusion chromatography revealed that the 48-kDa TNFR1 in human plasma did not fractionate with soluble proteins, but instead co-segregated with LDL particles on the basis of size. The 48-kDa TNFR1 in human plasma segregated independently from LDL particles by peak density, which demonstrates that TNFR1 exosome-like vesicles are distinct from LDL particles. Known exosome-associated proteins co-segregated with the HDL fraction of human plasma, which suggests that TNFR1 exosome-like vesicles are distinct from typical exosomes. This shows that human plasma contains 48-kDa TNFR1 exosome-like vesicles that fractionate with, but are distinct from, LDL particles, and display unique characteristics as compared to plasma- or endothelial cell-derived exosome-like vesicles (BBRC 2008 366: 579 - 584). V. The role of Toll-like receptor (TLR) signaling pathways that mediate TNFR1 release to the extracellular space was investigated. We found that poly (I:C), a viral double-stranded RNA (dsRNA) analog, selectively induces the cleavage and shedding of 34-kDa soluble TNFR1 ectodomains, but does not enhance the release of full-length 55-kDa TNFR1 within exosome-like vesicles from human airway epithelial cells. The poly (I:C)-mediated increases in sTNFR1 shedding are mediated via TLR3, whereas ligands for other toll-like receptors, including TLR4, TLR7 and NOD2, do not. Furthermore, we show that poly (I:C)-induced sTNFR1 release is mediated via two TLR3-TRIF-RIP1-dependent pathways. One pathway involves the Duox2-mediated generation of reactive oxygen species (ROS), while the second pathway is via caspase-mediated activation of apoptosis. We conclude that viral dsRNA-induced shedding of 34-kDa sTNFR1 ectodomains from human bronchial epithelial cells represents a novel mechanism by which innate immune responses to viral infections are modulated (J Immunol 2011;186:1180-1188).